Agrobacterium-mediated transformation of plants results in the integration of a T-strand within the genome of the plant cell. The T-strand contains gene expression cassettes that are made up of gene regulatory elements that have been precisely engineered to link a promoter to a gene of interest and 3′ untranslated region (UTR). The sequences are precisely engineered in relation to one another to optimally drive expression of the gene of interest to produce protein. The stability of the gene regulatory elements is crucial for the optimal expression of the gene of interest. Minor modification of the polynucleotide sequences that are contained within the T-strand can reduce or even eliminate expression of the gene of interest.
The Agrobacterium tumefaciens (LBA4404) strain is commonly used for integrating a T-strand within the genome of the plant cell. See Ooms, G., Hooykaas, P. J. J., Van Veen, R. J. M., Van Beelen, P., Regensburg-Tuienk, T. J. G., and R. A. Schilperoort (1982 “Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region.” Plasmid 7: 15-29; Hoekema, A., Hirsch, P. R., Hooykaas, P. J. J., and R. A. Schilperoort (1983) “A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid.” Nature 303:179-180; and, de Frammond, A. J., Barton K. A., and M-D. Chilton (1983) “Mini-Ti: a new vector strategy for plant genetic engineering”. Biotechnology 1: 262-269.
Despite the extensive use of A. tumefaciens (LBA4404) over the last thirty years, it has been observed that the plasmids transformed within this strain become unstable upon transformation within the strain. Gene regulatory elements, especially those elements that are repeated, have been observed to recombine within the A. tumefaciens (LBA4404) strain. This instability results in reduced plant transformation efficiency and the need to thoroughly screen potential transgenic plants for unaltered T-strand sequences. Given the instability of the plasmids transformed within this strain, a need exists for development of Agrobacterium tumefaciens (LBA4404) strains that do not possess recombination properties, and that can stably maintain a plasmid without rearrangements of the genetic elements located within the plasmid.
Thus, there remains a need for stains of Agrobacterium tumefaciens with improved plasmid stability. In particular, development for stains of Agrobacterium tumefaciens with deficiency in genetic recombination pathways would be desirable.